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Affinity Chromatography 13
Liquid chromatography is regarded as an indispensable tool in proteomics
allowing the discrimination of proteins by diverse principles based on reversephase,
ion exchange, size-exclusion, hydrophobic and affinity interactions (65).
The technique is potentially useful not only for the separation of specific groups
of proteins, but also for the exploration of post-translational modifications and
the study of protein–protein and protein–ligand interactions (66).
Furthermore, the use of affinity chromatography to enrich scarce proteins
or deplete over-abundant proteins is a powerful means of enhancing the
resolution and sensitivity in two-dimensional electrophoresis (2D-PAGE) or
mass spectrometry (MS) analysis. Isotope-encoded affinity tags may represent
a new tool for the analysis of complex mixtures of proteins in living systems
(67). Alternatively, element-encoded metal chelates may also prove helpful for
affinity chromatography, quantification and identification of tagged peptides
from complex mixtures by LC-MS/MS (68).
A significant development in affinity techniques for proteomics is the use of
fusion tags or proteins for expression and purification (69–71). A large choice
of systems is available for expression in bacterial hosts, with a further selection
amenable for eukaryotic cells. Amongst the most popular fusion partners for
molecular, structural and bioprocessing applications are the polyArg (72),
hexaHis-tag (73), glutathione-S-transferase (74) and maltose-binding protein
(75). Other less commonly employed expression tags include thioredoxin (76),
the Z-domain from Protein A (77), NusA (68), GB1 domain from Protein G (78)
and others (79). A recent comparison of the efficiency of eight elutable affinity
tags for the purification of proteins from E. coli, yeast, Drosophila and HeLa
extracts shows that none of these tags is universally superior for a particular
system because proteins do not naturally lend themselves to high throughput
analysis and they display diverse and individualistic physicochemical properties
(80). It was found that the His-tag provided good yields of tagged protein from
inexpensive, high capacity resins but with only moderate purity from E. coli
extracts and poor purification from the other extracts. Cellulose-binding protein
provided good purification from HeLa extracts. Consequently, affinity tags
are invaluable tools for structural and functional proteomics as well as being
used extensively in the expression and purification of proteins (81). Affinity
tags can have a positive impact on the yield, solubility and folding of their
complementary fusion partners. Combinatorial tagging might be the solution to
choosing the most appropriate partner in high throughput scenarios (70,81).
2.5. Resolution of Isoforms
Heterogeneity in proteins may arise due to variations in post-translational
modifications during the synthesis of a protein in native, recombinant or